The expression of the two ERO1-L isoforms therefore appears to be differently regulated, in the way that ERO1-La expression is mainly controlled by the cellular oxygen tension, whilst ERO
Trang 1The cellular oxygen tension regulates expression of the endoplasmic oxidoreductase ERO1-La
Bernhard Gess1, Karl-Heinz Hofbauer1, Roland H Wenger2, Christiane Lohaus3, Helmut E Meyer3
and Armin Kurtz1
1
Institut fu¨r Physiologie der Universita¨t Regensburg, Germany;2Carl-Ludwig-Institut fu¨r Physiologie der Universita¨t Leipzig, Germany;3Medizinisches Proteom-Center der Ruhr-Universita¨t, Bochum, Germany
The formation of disulfide bonds in the endoplasmic
reti-culum requires protein disulfide isomerase (PDI) and
endoplasmic reticulum oxidoreductin 1 (ERO1) that
reoxi-dizes PDI We report here that the expression of the rat,
mouse and human homologues of ERO1-Like protein a but
not of the isoform ERO1-Lb are stimulated by hypoxia in
rats vivo and in rat, mouse and human cell cultures The
temporal pattern of hypoxic ERO1-La induction is very
similar to that of genes triggered by the hypoxia inducible
transcription factor (HIF-1) and is characteristically
mim-icked by cobalt and by deferoxamine, but is absent in cells
with a defective aryl hydrocarbon receptor translocator
(ARNT, HIF-1b) We speculate from these findings that
the expression of ERO1-La is probably regulated via the
HIF-pathway and thus belongs to the family of classic oxygen regulated genes Activation of the unfolded protein response (UPR) by tunicamycin, on the other hand, strongly induced ERO1-Lb and more moderately ERO1-La expres-sion The expression of the two ERO1-L isoforms therefore appears to be differently regulated, in the way that ERO1-La expression is mainly controlled by the cellular oxygen tension, whilst ERO1-Lb is triggered mainly by UPR The physiological meaning of the oxygen regulation of ERO1-La expression likely is to maintain the transfer rate of oxidizing equivalents to PDI in situations of an altered cellular redox state induced by changes of the cellular oxygen tension Keywords: hypoxia; HIF; protein folding; UPR; PDI
Formation of disulfide bonds is an essential event for the
correct folding of proteins in the endoplasmic reticulum
It is well known that this process is catalyzed by protein
disulfide-isomerase (PDI) [1] Until a few a years ago,
however, it remained unclear how PDI is reoxidized in this
reaction [2] It was the discovery of the ERO1
(endoplas-mic reticulum oxidoreductin) protein in yeast [3,4] which
provided evidence that this protein is essential to transfer
oxidizing equivalents to PDI [5] It turned out that ERO1
is a highly conserved endoplasmic protein and for humans
and mouse two ERO1-Like proteins have meanwhile been
identified, termed ERO1-La [6] and -1b [7] The ERO1
proteins are probably flavoproteins [8] that covalently bind
to PDI [9], what explains their function to transfer
oxidizing equivalents to PDI ERO1-La and -Lb display
different tissue distributions [7], and moreover, appear to
be differently regulated in their expression Thus, mainly
ERO1-L b transcripts are induced in the course of
unfolded protein response [7] In this pathway
accumula-tion of misfolded proteins in the endoplasmic reticulum
induces the expression of a number of proteins including those involved in the correct folding of proteins such as chaperones [10] How the expression of the ERO1-La protein is regulated is yet unknown Analyzing the protein expression pattern of a rat vascular smooth muscle cell line,
we now found that a ERO1-Like protein highly homo-logous to mouse and human ERO1-La is strongly upregulated during cellular hypoxia This study therefore aimed to characterize the effects of low oxygen tension on ERO1-L(a) expression
Materials and methods Cell cultures
Rat aortic vascular smooth muscle cells (A7r5) from BDXI rats (ATCC CRL 1444) were cultured in 75 cm2 flasks (Sarstedt) with 15 mL Dulbecco’s minimal essential medium (MEM) containing 10% fetal bovine serum and penicillin/streptomycin (10 U/10 lgÆmL)1, Biochrom), kept
in room air with 10% CO2at 37C Medium was changed every second day and cells were confluent on day 7–10 after splitting which was achieved with trypsin-EDTA for 5 min
at 37C For the experiments, cell cultures (triplicates) were incubated at room air (21% O2i.e normoxia) or 1%
O2 or 0.5% O2 (i.e hypoxia) for up to 12 h Additional culture dishes were incubated at 21% O2 with either cobaltous chloride (100 lmolÆL)1) or with deferoxamine (100 lmolÆL)1) for 12 h
To induce the unfolded protein response, A7r5 cells were incubated with 5 lgÆmL)1tunicamycin for 4.5, 8, 12 and
24 h
Correspondence to A Kurtz, Institut fu¨r Physiologie,
Universita¨t Regensburg, D-93040 Regensburg, Germany.
Fax: + 49 941 943 4315, Tel.: + 49 941 943 2980,
E-mail: armin.kurtz@vkl.uni-regensburg.de
Abbreviations: PDI, protein disulfide isomerase; ERO, endoplasmic
reticulum oxidoreductin; ERO1-L, ERO1-Like protein; HIF, hypoxia
inducible transcription factor; ARNT, aryl hydrocarbon receptor
translocator; UPR, unfolded protein response.
(Received 14 February 2003, revised 18 March 2003,
accepted 25 March 2003)
Trang 2Mouse hepatoma Hepa1 cells, and their subclone
Hepa1C4, which produces defective aryl hydrocarbon
receptor (ARNT, HIF-1b) [11] due to a point mutation
[12] rendering the cells unable to form active hypoxia
inducible factor HIF [13], were grown under the above
mentioned conditions For the experiments the cells were
incubated either at 0.5% O2(i.e hypoxia) or at 21% O2with
deferoxamine (100 lmolÆL)1) for 4.5 and 12 h
Human hepatoma HepG2 cells (used at 50% confluency)
and the mouse renin secreting cell line As4.1 [14] were also
grown under the above mentioned conditions The cells
were incubated either at 0.5% O2(i.e hypoxia) or at 21%
O2with deferoxamine (100 lmolÆL)1) for 4.5 h
In vivo experiments
All experiments were conducted in accordance with the
National Institutes of Health Guide for the Use of
Laboratory Animals and the German Law on the
protec-tion of Animals Male Sprague–Dawley rats (200–250 g)
that had free access to food and water were used for the
experiments and treated in the following way (a): in the
control group, animals received no treatment (n¼ 6) (b); in
the hypoxia group, the animals were placed in a gas-tight
box that was supplied continuously with a gas mixture of
8% O2-92%N2for 6 h (n¼ 6) (c); in the carbon monoxide
group, the animals were placed in a gas-tight box that
was supplied continuously with room-air plus 0.1% CO for
6 h (n¼ 6); and [4] for cobalt treatment, the rats were
subcutaneously injected with cobalt chloride (60 mgÆkg)1),
and the animals were killed 6 h later (n¼ 6) At the end of
the experiments, the animals were killed by decapitation
Aortas, brains, hearts, kidneys, livers and lungs were
removed quickly, weighed, and rapidly frozen in liquid
nitrogen All organs were stored at)80 C until isolation of
protein and total RNA
Preparation of protein samples
After removal of cell culture medium, cells were washed
three times with ice-cold NaCl/Piand then scraped off in
lysis buffer (300 lL per 75 cm2 flask) consisting of
7 molÆL)1urea, 2 molÆL)1thiourea, 2% Chaps, 1%
dithio-threitol, Pharmalyte pH 3–10 L (Pharmacia, Uppsala,
Sweden), supplemented with protease inhibitors
(com-plete, Boehringer Mannheim, Germany) The material
was then homogenized with an Ultraturrax (3· 10 s) and
further sonicated for 3· 10 s The homogenate was then
allowed to stand at room temperature for 60 min prior to
ultracentrifugation at 80 000 g at 15C for 1 h Aliquots of
the clear supernatant were frozen in liquid nitrogen and
stored at)80 C For determination of the protein
concen-tration, protein was precipitated with 10% trichloroacetic
acid in acetone and resuspended in 0.1MNaOH Protein
concentration was then determined with the Bio-Rad
protein assay (BIO-RAD, Int)
Two-dimensional PAGE
Protein (150 lg, for silverstained gels) and 600 lg protein
(for Coomassie-Blue staining) were loaded for each sample
onto the first dimension strips A linear immobilized pH
gradient (pH 5.0–6.0 IPG 18 cm; Pharmacia, Uppsala, Sweden) was used as the first dimension Hydration of gel strips and sample application was performed at 50 V for
15 h For protein separation a step voltage protocol was applied (1 h 150 V, 3 h 500 V, 1 h 1000 V, gradient
to 8000 V within 0.5 h) A total volt-hour product of
60 kVh was used for 150 lg protein and 110 kVh for
600 lg protein Afterwards the stripes were incubated in
50 mmolÆL)1Tris/HCl (pH 6.8), urea 6 molÆL)1, glycerol 30%, dithiothreitol 65 mmolÆL)1, 2% SDS for 20 min at room temperature followed by incubation in 50 mmolÆL)1 Tris/HCl (pH 8.8), urea 6 molÆL)1, glycerol 30%, iodo-acteamide 140 mmolÆL)1, 2% SDS for another 20 min For the second dimension, a vertical gradient slab gel of 8%)18% acrylamide was used and SDS/PAGE was performed at 8 mA per gel at 13C for 4 h followed by
30 mA for 12 h At the end of the second dimension, the gels were removed from the glass plates
Staining of two-dimensional PAGE The gels were fixed and stained with silver according to standard protocols [15] The gels were then scanned (Image Scanner Sharp JX-330, Amersham Biosciences) and ana-lysed with theIMAGE3.1 analysis software package (Amer-sham Bioscience) Each spot was matched from one gel
to another and the relative volume of matched spots was compared For preparative protein analysis higher amounts
of protein were loaded for two-dimensional PAGE and the protein spots were then stained with colloidal Coomassie-Blue
Protein sequence analysis Coomassie-Blue stained spots were excised from the gels and were subjected to ESI-MS analysis [16] Sequences obtained with ESI-MS analysis were then compared with the mouse and rat subset of the NCBInr.fasta protein database
cDNA cloning From the protein sequence of the obtained peptides the coding DNA sequence was obtained with database stand-ard programs A pair of sense primer 5¢-CGGGATCC TGCGAGCTACAAGTATTC-3¢ and antisense down-stream primer 5¢-GGAATTCTCCACATACTCAGCA TCG-3¢ was then used for standard RT-PCR cloning of a cDNA fragment of the sequenced protein A 192-bp cDNA fragment with the sequence: 5¢-tccacatactcagcatcgggggactg tatgtcatcaacttcacagaagctgtctgaagaatcatcgtgtttcgtccactgaaga acagccttctgggtctcctcactcagagattcgtccactgctccgagccgctcagcct gctcacactcctcaaggaggttggcttccttggaatacttgtagctcgca-3¢ was obtained This sequence was then further used for sequence comparisons and to generate a cRNA probe for RNase protection
RNA isolation Total RNA was extracted from freshly harvested cells and from frozen tissues according to the protocol of Chom-czynski and Sacchi [17]
Trang 3RNase protection assay of ERO1-L(a), adrenomedullin
(ADM) and b-actin mRNA
ERO1-L(a), ADM and b-actin mRNA levels were
meas-ured by RNase protection assay as described previously
[18] In brief, radiolabelled antisense cRNA probes were
synthesized by in vitro transcription of plasmid vectors
carried subcloned cDNA fragments for ERO1-L, ADM
and b-actin with SP6 polymerase (Promega) in the presence
of [a-32P]GTP (Amersham) Labeled cRNA probes were
hybridized with total RNA at 60C for 16 h, then digested
with RNase A/T1 at room temperature for 30 min and
proteinase K at 37C for 30 min After phenol/chloroform
extraction and ethanol precipitation, the protected RNA
hybrids were separated by electrophoresis on 8%
polyacryl-amide gels After drying the gels, the amount of
radio-activity was assessed by an Instant Imager (Packard) in
counts per minute (c.p.m.) and autoradiography was
performed at)80 C for 1 day Results were expressed as
in proportion to b-actin mRNA as internal standard
Real time PCR analysis of mouse and human ERO1-La
and ERO1-Lb mRNA and b-actin mRNA
Real time PCR was performed in a Light Cycler (Roche,
Germany) All PCRexperiments were performed using the
Light Cycler DNA Master SYBRGreen I kit provided by
Roche Molecular Biochemicals (Mannheim, Germany)
Each reaction (20 lL) contained 2 lL cDNA, 3.0 mM
MgCl2, 1 pmol of each primer and 2 lL of Fast Starter
Mix (containing buffer, dNTPs, SYBRGreen and hotstart
Taq polymerase) The following primers were used For
human ERO1-La (gi|6272556); sense primer: 5¢-CGGGAT
CCTGATGAAGTTCCTGATGG-3¢, antisense primer:
5¢-GGAATTCGTCTGTGGCTTAAAACAG-3¢ For
human ERO1-Lb (gi|9716556); sense primer: 5¢-CGGGAT
CCCTGGGCAAGATATGATGA-3¢, antisense primer:
5¢-GGAATTCATTGATGCTAGCATGAAG-3¢ For
mouse ERO1-La (gi|15718668); sense primer: 5¢-CGGGA
TCCTGCGAGCTACAAGTATTC-3¢, antisense primer:
5¢-GGAATTCGCCACATACTCAGCATCg-3¢ For
mouse ERO1-Lb (gi|19744822); sense primer: 5¢-CGG
GATCCCTTTTGTGAACTTGATGA-3¢, antisense
pri-mer: 5¢-GGAATTCAGCCACGTATAGAATGAt-3¢
For mouse and human b-actin (gi|6671508); sense primer:
5¢-CGGGATCCCCGCCCTAGGCACCAGGGTG-3¢,
antisense primer: 5¢-GGAATTCGGCTGGGGTGTTGA
AGGTCTCAAA-3¢
The amplification program consisted of 1 cycle at 95C
for 10 min, followed by 40 cycles with a denaturing phase at
95C for 15 s, an annealing phase of 5 s at 60 C and a
elongation phase at 72C for 15 s A melting curve analysis
was performed after amplification to verify the accuracy of
the amplicon For verification of the correct amplification,
PCRproducts were analyzed on an ethidium bromide
stained 2% agarose gel
In each real-time-PCRrun for ERO1-L and for b-actin a
calibration curve was included, that was generated from
serial dilutions (1 : 1, 1 : 10, 1 : 100, 1 : 1000) of a cDNA
generated from the pooled RNA of the normoxic (control)
cultures (time 0) of the respective experimental series
(standard cDNA) Analysis of the individual unknowns
therefore yielded values relative to this pool Data are presented as the relative ERO1-L mRNA/b-actin mRNA ratio The ERO1-L mRNA/b-actin mRNA ratio of the standard (pool) cDNA was set to 1.0 (i.e time 0)
Statistics Levels of significance between groups were calculated using ANOVAtest followed by Bonferoni’s reduction for multiple comparisons P < 0.05 was considered significant Results
Screening the rat vascular smooth muscle cell line A7r5 for hypoxia induced proteins by 2D-electrophoresis revealed a highly reproducible and marked (about 20-fold) upregulated abundance of a protein with an pI of around pH 5.7 and
an apparent molecular mass of 58 kDaA on SDS/PAGE (Fig 1) By ESI-MS tryptic peptides were identified that covered 45.9% of the aminoacid sequence of the mouse ERO1-like protein, which consists of a total of 464 amino acids (gi|7657067) Based on the sequenced peptides a
Fig 1 2D-electrophoresis of proteins isolated from the rat vascular smooth muscle cell line, A7r5 kept at either 21% O 2 (A) or 1% O 2 (B) for 12 h Note the upregulation of the indicated protein spot.
Trang 4cDNA fragment was cloned by RT-PCR standard
tech-niques The resulting 192 bp cDNA sequence shared a
100% homology with rat ERO1-1(gi|18250365), 88%
homology with mouse ERO1-La (gi|15718668), 85%
homology with human ERO1-La (gi|7021225), but no
significant homology with human ERO1-Lb (gi|9845248)
or mouse ERO1-Lb (gi|19744822)
It was concluded therefore that the cloned cDNA was rat
ERO1-L(a) cDNA and the hypoxia induced protein was rat
ERO1-L(a) [rER O1-L(a)] The cloned cDNA was then
used to generate cRNA probes for quantification of
rERO1-L(a) mRNA by RNAse protection
It turned out that the abundance of rERO1-L(a) mR NA
in A7r5 cells at high oxygen tensions (21% O2) was rather
low, but increased strongly (20-fold) with a characteristic
time pattern and reached a stable plateau level after
exposure of the cells to low oxygen tensions (1% O2)
(Fig 2, upper panel)
A next set of experiments was designed to test for the
in vivorelevance of the findings obtained with A7r5 cells
For this goal rats were exposed either to room atmosphere
(21% O2) or to a low inspiratory oxygen tension (8% O2)
and rERO1-1(a) mRNA was semiquantitated by RNAse
protection in the different organs As shown in Table 1
rERO1-L(a) mRNA was upregulated by hypoxia in all
organs examined, except the brain, in which only a marginal
increase was found To determine whether the upregulation
of rERO1-L(a) was not only related to a fall of the arterial oxygen tension but more generally to a fall of cellular oxygen tension, we also examined the effect of carbon monoxide (CO) inhalation [0.1%] 0.1% CO inhibits oxygen transport by hemoglobin by about 50% and thus diminishes oxygen delivery to the tissues without changing arterial oxygen tension Depending on the rate of tissue oxygen consumption CO will therefore lower tissue oxygen tension
It turned out that also CO clearly stimulated rERO1-L(a) mRNA levels in the different rat organs, with the exception
of the lung, in which tissue oxygen tensions are directly related to inspiratory oxygen tensions rather than to the oxygen carrying capacity of the blood (Table 1) Thus, the failure of CO to stimulate rERO1-L(a) expression in the lung, can be taken as an argument that CO did not itself increase rERO1-L(a) expression rERO1-L(a) in vivo was also stimulated by the divalent cation cobalt, that was subcutaneously administered [Table 1]
The temporal pattern of rERO1-L(a) mR NA in rat A7r5 cells was very similar to that of classic oxygen regulated genes, such as adrenomedullin (ADM) (Fig 2, lower panel), the expression of which is triggered by the hypoxia inducible transcription factor HIF-1 [19] In addition, rERO1-L(a) mRNA was, like ADM mRNA, upregulated by the divalent cation cobalt (100 lmolÆL)1) and by the iron chelator deferoxamine (100 lmolÆL)1) (Fig 3)
Hypoxia and deferoxamine also increased ERO1-La mRNA in the mouse hepatoma cell line Hepa1 (Fig 4), suggesting a species independent stimulatory effect of hypoxia on ERO1-La gene expression In contrast, in the mutant cell line Hepa1C4, which is unable to generate active HIF [13], hypoxia and deferoxamine failed to increase ERO1-La mRNA (Fig 4) within the first five hours Only after 12 h of hypoxia or incubation with deferoxamine ERO1-La mRNA increased moderately
Using Hepa1 cells we also examined the effect of hypoxia and desferoxamine on the abundance of ERO1-Lb mRNA
As shown in Fig 5 there was no change of ERO1-Lb mRNA after 4.5 h, when ERO1-La mRNA levels had already clearly increased After 12 h of hypoxia ERO1-Lb mRNA was moderately elevated In view of the different temporal response of ERO1-La and ERO1-Lb mRNA to hypoxia in mouse Hepa1 cells, we analyzed the early hypoxic response also in the mouse renal renin secreting As4.1 cell line [14] and in the human hepatoma Hep G2 cell
Fig 2 Time course of rERO1-L mRNA (upper panel) and of
adreno-medullin mRNA (lower panel) in A7r5 cells after exposure of the cells to
1% O 2 Data are means ± SEM of five experiments *Indicates
P < 0.05 hypoxia (1% O ) vs normoxia (21% O ).
Table 1 Effect of hypoxia (8% O 2 ), carbon monoxide (0.1%) inhala-tion and of administrainhala-tion of 60 mgÆkg)1 cobaltous chloride on ERO1-La mRNA in various rat tissues Results are presented as ratio ERO1-L(a) mR NA/b-actin mRNA · 10 2
Data are means ± SEM
of 4–6 animals *Indicates P < 0.05 vs 21% O 2
Organ 21% O 2 8% O 2 0.1% CO
Cobaltous chloride (60 mgÆkg)1) Aorta 7 ± 1 14 ± 3* 16 ± 4* 11 ± 4 Brain 5.6 ± 1.6 6.5 ± 0.5 11.2 ± 1.4* 9.6 ± 2.1* Heart 8 ± 1 15 ± 2* 23 ± 4* 19 ± 5* Kidney 12 ± 4 57 ± 21* 31 ± 8* 25 ± 4* Liver 20 ± 8 110 ± 28* 350 ± 30* 230 ± 30* Lung 1.8 ± 0.3 2.9 ± 0.5* 1.9 ± 0.2 3.8 ± 1.1*
Trang 5line It turned out that 4.5 h of hypoxia induced ERO1-La
but not ERO1-Lb mRNA (Fig 6) Similar results were
obtained for the effect of deferoxamine
As a differential regulation of ERO1-La and ERO1-Lb
mRNA expression has been reported previously, in the way
that the unfolded protein response (UPR) pathway
prefer-entially induces ERO1-Lb mRNA expression [7], we aimed
to examine this concept in our model of mouse As4.1 cells
We found, that tunicamycin (5 lgÆmL)1), which induces the
UPR, increased ERO1-La and ERO1-Lb mRNA about
fourfold after 4.5 h of incubation Whilst ERO1-Lb mRNA
further increased to a plateau 12-fold above control,
declined ERO1-La mRNA after prolonged incubation to
reach a plateau twofold over control (Fig 7)
Discussion
Correct protein folding in the endoplasmic reticulum
essentially requires the activity of the protein disulfide
isomerase PDI, which in turn is dependent on the delivery
of oxidizing equivalents by endoplasmic oxidoreductase
ERO1, which occurs in an La- and in a Lb-isoform in
mammals ERO1-L isoforms in conjunction with PDI
therefore fulfil chaperone function It is well known that a
variety of endoplasmic proteins with chaperone function are
induced by energy depletion caused by severe cellular
hypoxia (anoxia) or by glucose deprivation [20] It is
thought that the expression of these proteins in response to anoxia is triggered by the unfolded protein response (UPR) which regulates the activity of chaperone genes [21] and leads to attenuation of protein synthesis via the activation of the endoplasmic reticulum kinase PERK [22] Unfolding or misfolding of proteins in the endoplasmic reticulum during anoxia probably results from ATP depletion and also from changes of redox potentials In consequence, yeast ERO1 [3] and ERO1-L b in human tissues [7] are also stimulated by UPR Interestingly, ERO1-L a appears to be less affected by UPR[7] suggesting that ERO1-L a is differently regulated
in its expression
Our data now indicate that the expression of the rat, mouse and human isoform of ERO1-L(a) is strongly upregulated following a decrease in the cellular oxygen tension Apparently, this phenomenon appears to be of major relevance also under in vivo conditions under which rERO1-L(a) expression is also markedly increased during hypoxia Our data also show that not only arterial hypoxia but also a reduction of the oxygen carrying capacity of the blood (by CO inhalation) stimulates rERO1-L(a) gene expression in various tissues
Our data provide several lines of evidence to suggest that the expression ERO1-La is probably triggered by the hypoxia-inducible transcription factor (HIF-1)
Fig 3 rERO1-L(a) mRNA (upper panel) and adrenomedullin mRNA
(lower panel) in A7r5 cells after exposure to 0.5% O 2 or to cobaltous
chloride (100 lmolÆL)1) or deferoxamine (100 lmolÆL)1) for 12 h at
21% O 2 Data are means ± SEM of five experiments each *Indicates
P < 0.05 vs control (21% O 2 ).
Fig 4 Mouse ERO1-La mRNA in Hepa1 (upper panel) and in Hepa1C4 cells (lower panel) after exposure to hypoxia (0.5% O 2 ) (100 lmolÆL)1) or to deferoxamine (100 lmolÆL)1) at 21% O 2 mRNA was semiquantitated by real-time PCR Data are means ± SEM of five experiments each *Indicates P < 0.05 vs control (21% O 2 ).
Trang 6HIF-1 is a heterodimer consisting of an a- and a
b-subunit [23] HIF-1a stability is regulated by the cellular
oxygen tension, in the way that an oxygen/iron dependent
prolyl-hydroxylation leads to increased ubiquitinylation
and finally proteasomal degradation of HIF-1a [24,25] In
consequence, a decrease of prolyl-hydroxylase activity by
low oxygen tensions, by iron chelation or by cobalt increase
HIF-1a protein levels and therefore the activity of the
HIF-1 transcription factor [26]
The temporal pattern of the induction of rERO1-L(a)
expression by hypoxia in vitro is very similar to HIF-1
regulated genes, such as adrenomedullin [19] Moreover, the
effect of hypoxia on ERO1-La gene expression can be
mimicked in a very characteristic fashion by cobalt and by
the iron chelator deferoxamine, which do not change
cellular oxygen tension but increase HIF-1a and therefore
stimulate HIF-1 activity [27,28] Finally, the early
stimula-tion of ERO-1a gene expression was absent in a cell line
with a functional mutation in the HIF-1b gene, which
causes an inability to form active HIF [13] The moderate
increase of ERO-1a gene expression in HIF deficient cells
after prolonged hypoxia is probably explained by unfolded
protein response pathway, which is evoked by prolonged
hypoxia and which itself moderately triggers ERO1-La
gene expression as seen in this study [Fig 7]
In contrast to ERO-1a gene expression, ERO1-Lb mRNA was not upregulated by acute hypoxia in the mouse and human cell lines, suggesting that hypoxia per se is not a major trigger for ER O1-Lb gene expression The moderate
of increase of ERO1-Lb mRNA by prolonged hypoxia may
be again explained by the induction of the unfolded protein response, what would well fit with the concept that the UPR mainly triggers the ERO1-Lb gene [7]
The conclusion that EROl-La but not ERO1-Lb is triggered by HIF-1 is indirectely supported by the occurence
of the most common active HIF-binding consensus sequence ACGTG in the ERO1-L gene promotors Thus, rat, mouse and human EROl-La contain two, two and one ACGTC motifs in CpG islands in the 5¢-promoter region, respectively, whilst ERO1-Lb does not contain this motif
in GpC islands
HIF-1 regulated genes identified so far encode proteins that mainly serve to match the cellular energy deficit resulting from insufficient oxygen supply [29] Thus, glucose transporters and key enzymes of the glycolytic pathway are regulated by HIF-1 and are upregulated during hypoxia Also secreted proteins such as erythropoietin which stimu-lates red cell formation (and thus increases the oxygen carrying capacity of the blood) or vascular endothelial growth factor (VEGF), which induces capillary formation,
Fig 6 ERO1-La and ERO1-Lb mRNA in mouse As4.1 cells (upper panel) and in human HepG2 cells (lower panel) after exposure to hypoxia (0.5% O 2 ) (100 lmolÆL)1) or to deferoxamine (100 lmolÆL)1) at 21%
O 2 after 4.5 h of incubation mRNA was semiquantitated by real-time PCR Data are means ± SEM of five experiments each *Indicates
P < 0.05 vs control (21% O 2 ).
Fig 5 Mouse ERO1-La (upper panel) and ERO1-Lb mRNA (lower
panel) in Hepa1 (upper panel) cells (lower panel) after exposure to
hypoxia (0.5% O 2 ) or to deferoxamine (100 lmolÆL)1) at 21% O 2
mRNA was semiquantitated by real-time PCR Data are means ±
SEM of five experiments each *Indicates P < 0.05 vs control
(21% O 2 ).
Trang 7or adrenomedullin (ADM), which causes vasodilation, are
stimulated by HIF-1 in response to hypoxia (reviewed in
[29])
With the regulation of proteins that are involved in
correct folding of proteins in the endoplasmic reticulum,
HIF-1 would aquire a new responsibility for cellular
function (Fig 8) A regulation of ERO1-La production
by HIF-1 means that chaperone formation during hypoxia
is uncoupled from energy depletion (which initiates the
UPR), and thus allows a counterregulation in situations in
which the cellular redox state is already altered whilst the
energy state is still normal A number of endo- or paracrine
signals involved in the hypoxia defense such as for example
erythropoietin [30], VEGF [31] or ADM [32] in fact contain
disulfide bonds that are indispensable for their biological
function Problems with disulfide bond formation during a
fall of the oxygen tension may arise from the change of the
redox potential of the cell, which impairs the flow rate of
oxidizing equivalents from ERO1-L to PDI Under
redu-cing conditions PDI would actually catalyze the reduction
of protein disulfides [1] The relevance of PDI in this context
was underlined previously by the finding that
overexpres-sion of PDI attenuated the loss of cell viability induced by
hypoxia in a neuroblastoma cell line [33] As ERO1-La
exists as a collection of oxidized and reduced forms [9]
increasing the total number of ERO1-La molecules during
hypoxia would therefore compensate for the diminuation of
the redox gradient and maintain a constant flow of oxidizing equivalents to PDI over a broad range of cellular oxygen tension
The oxygen regulation of ERO1-La expression appears
to be part of a more general network in which the expression
of chaperones is regulated by the oxygen tension through HIF-1 Thus, it was shown previously that hypoxia increases the expression of PDI itself in brain cells in vitro and in vivo [33], although it was not further examined in that study as to whether the upregulation of PDI was mediated
by UPRor by the HIF-1 pathway PDI also serves as the b-subunit of the collagen prolyl-4-hydroxylase, which is a heterotetramer consisting of 2a and 2b subunits [34] It was reported previously for cultured fibroblasts that hypoxia induces the expression of a-subunit of the collagen prolyl-4-hydroxylase (I) through the HIF-1 pathway [35] All together, our findings suggest that a fall of the cellular oxygen tension compensatorily increases the expression of a protein that is required to transfer oxidizing equivalents to PDI, and is therefore required for correct protein folding in the endoplasmic reticulum
Acknowledgements
The authors thank K-H Go¨tz for doing the artwork and Vladimir Todorov for helpful discussions.
References
1 Fassio, A & Sitia, R (2002) Formation, isomerisation and reduction of disulphide bonds during protein quality control in the endoplasmic reticulum Histochem Cell Biol 117, 151–157.
2 Freedman, R B., Dunn, A.D & R uddock, L.W (1998) Protein folding: a missing redox link in the endoplasmic reticulum Curr Biol 18, R468–R470.
Fig 8 Summary of the regulation of ERO1-L expression by the oxygen tension and by the unfolded protein response Abbreviations: pO 2 , cel-lular oxygen tension; ATP, concentration of adenosine triphosphate; ERendoplasmic reticulum; UPR , unfolded protein response; PDI, protein disulfide isomerase.
Fig 7 ERO1-La and ERO1-Lb mRNA in mouse As4.1 cells after
incubation with tunicamycin (5 lgÆmL)1) at 21% O 2 mRNA was
semiquantitated by real-time PCR Data are means ± SEM of five
experiments each *Indicates P < 0.05 vs control (21% O 2 ).
Trang 83 Frand, A.R & Kaiser, C.A (1998) The ERO1 gene of yeast is
required for oxidation of protein dithiols in the endoplasmic
reticulum Mol Cell 1, 161–170.
4 Pollard, M.G., Travers, K.J & Weissman, J.S (1998) ERO1p: a
novel and ubiquitous protein with an essential role in oxidative
protein folding in the endoplasmic reticulum Mol Cell 1, 171–
182.
5 Frand, A.R & Kaiser, C.A (1999) ERO1p oxidizes protein
disulfide isomerase in a pathway for disulfide bond formation in
the endoplasmic reticulum Mol Cell 4, 469–477.
6 Cabibbo, A., Pagani, M., Fabbri, M., Rocchi, M., Farmery,
M.R., Bulleid, N.J & Sitia, R (2000) ERO1-L, a human protein
that favors disulfide bond formation in the endoplasmic reticulum.
J Biol Chem 275, 4827–4833.
7 Pagani, M., Fabbri, M., Benedetti, C., Fassio, A., Pilati, S.,
Bul-leid, N.J., Cabbibo, A & Sitia, R (2000) Endoplasmic reticulum
oxidoreductin 1-l beta (ERO1-L beta), a human gene induced in
the course of the unfolded protein response J Biol Chem 275,
23685–23692.
8 Tu, B.P., Ho-Schleyer, S.C., Travers, K.J & Weissman, J.S (2000)
Biochemical basis of oxidative protein folding in the endoplasmic
reticulum Science 24, 1571–1574.
9 Benham, A.M., Cabibbo, A., Fassio, A., Bulleid, N., Sitia, R &
Braakman, I (2000) The CXXCXXC motif determines the
fold-ing, structure and stability of human ERO1-L alpha EMBO J 19,
4493–4502.
10 Ma, Y & Hendershot, L.M (2001) The unfolding tale of the
unfolded protein response Cell 107, 827–830.
11 Cuthill, S & Poellinger, L (1988) DNA binding properties of
dioxin receptors in wild-type and mutant mouse hepatoma cells.
Biochemistry 27, 2978–2982.
12 Numayama-Tsuruta, K., Kobayashi, A., Sogawa, K &
Fujii-Kuriyama, Y (1997) A point mutation responsible for defective
function of the aryl-hydrocarbon-receptor nuclear translocator in
mutant Hepa-1c1c7 cells Eur J Biochem 246, 486–495.
13 Gassmann, M., Kvietikova, I., Rolfs, A & Wenger, R.H (1997)
Oxygen- and dioxin-regulated gene expression in mouse hepatoma
cells Kidney Int 51, 567–574.
14 Sigmund, C.D., Okuyama, K., Ingelfinger, J., Jones, C.A.,
Mul-lins, J.J., Kane, C., Kim, U., Wu, C.Z., Kenny, L., R ustum, Y.,
Dzau, V.J & Gross, K.W (1990) Isolation and characterization of
renin expressing cell lines from transgenic mice containing a
renin-promoter viral oncogene fusion construct J Biol Chem 265,
19916–19922.
15 Blum, H., Beier, H & Gross, H.J (1989) The expression of the
TMV-specific 30-kDa protein in tobacco protoplasts is strongly
and selectively enhanced by actinomycin Virology 169, 51–61.
16 Sickmann, A., Marcus, K., Scha¨fer, H., Butt-Do¨rje, E., Lehr, S.,
Herkner, A., Suer, S., Bahr, I & Meyer, H.E (2001) Identification
of post-translationally modified proteins in proteome studies.
Electrophoresis 22, 1669–1676.
17 Chomczynski, P & Sacchi, N (1987) Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction Anal Biochem 162, 156–115.
18 Hofbauer, K.H., Jensen, B.L., Kurtz, A & Sandner, P (2000)
Tissue hypoxygenation activates the adrenomedullin system in
vivo Am J.Physiol 278, R513–R519.
19 Cormier-Regard, S., Nguyen, S.V & Claycomb, W.C (1998)
Adrenomedullin gene expression is developmentally regulated and
induced by hypoxia in rat ventricular cardiac myocytes J Biol.
Chem 273, 17787–17792.
20 Lee, A.S (2001) The glucose-regulated proteins: stress induction
and clinical applications Trends Biochem Sci 26, 504–510.
21 Heacock, C.S & Sutherland, R.M (1990) Enhanced synthesis of stress proteins caused by hypoxia and relation to altered cell growth and metabolism Br J.Cancer 62, 217–225.
22 Koumenis, C., Naczki, C., Koritzinsky, M., Rastani, S., Diehl, A., Sonenberg, N., Koromilas, A & Wouters, B.G (2002) Regulation
of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIFalpha Mol Cell Biol 22, 7405–7416.
23 Wang, G.L & Semenza, G.L (1995) Purification and characteri-zation of hypoxia-inducible factor 1 J Biol Chem 270, 1230– 1237.
24 Jaakkola, P., Mole, D.R., Tian, Y.M., Wilson, M.I., Gielbert, J., Gaskell, S.J., Kriegsheim, A.V., Hebestreit, H.F., Mukherji, M., Schofield, C.J., Maxwell, P.H., Pugh, C.W & Ratcliffe, P.J (2001) Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O 2 -regulated prolyl hydroxylation Science 292, 468–472.
25 Bruick, R.K & McKnight, S.L (2001) A conserved family of prolyl-4-hydroxylases that modify HIF Science 294, 1337–1340.
26 Epstein, A.C., Gleadle, J.M., MsNeill, L.A., Hewitson, K.S., O’Rourke, J., Mole, D.R., Mukherji, M., Metzen, F., Wilson, M.I., Dhanda, A., Tian, Y.M., Masson, N., Mailton, D.L., Jaa-kola, P., Barstead, R., Hodgkin, J., Maxwell, P.H., Pugh, C.W., Schofield, C.J & Ratcliffe, P.J.C (2001) elegans EGL-9 and mammalian homologs define a family of dioxygenases that regu-late HIF by prolyl hydroxylation Cell 107, 43–54.
27 Wang, G.L., Jiang, B.H & Semenza, G.L (1995) Effect of protein kinase and phosphatase inhibitors on expression of hypoxia-inducible factor 1 Biochem Biophys Res Commun 216, 669–675.
28 Jiang, B.H., Zheng, J.Z., Leung, S.W., R oe, R & Semenza, G.L (1997) Transactivation and inhibitory domains of hypoxia-inducible factor 1alpha Modulation of transcriptional activity by oxygen tension J Biol Chem 272, 19253–19260.
29 Semenza, G.L (1999) Regulation of mammalian O 2 homeostasis
by hypoxia-inducible factor 1 Annu Rev Cell Dev Biol 15, 551–578.
30 Shimizu, T., Miyake, T., Pilch, A.M., Mantel, C & Rhy, M.J Jr (1986) Biochemical properties of human urinary megakaryocyte colony-stimulating factor and erythropoietin: the role of sulf-hydryl Groups and disulfide bonds Exp Cell Res 54, 281–286.
31 Potgens, A.J., Lubsen, N.H., van Altena, M.C., Vermeulen, R., Bakker, A., Schoenmakers, J.G., Ruiter, D.J & de Waal, R.M (1994) Covalent dimerization of vascular permeability factor/ vascular endothelial growth factor is essential for its biological activity Evidence from Cys to Ser mutations J Biol Chem 269, 32879–32885.
32 Kitamura, K., Matsui, E., Kato, J., Katoh, F., Kita Tsuji, T., Kangawa, K & Eto, T (2001) Adrenomedullin (11–26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla Peptides 22, 1713–1718.
33 Tanaka, S., Uehara, T & Nomura, Y (2000) Up-regulation of protein-disulfide isomerase in response to hypoxia/brain ischemia and its protective effect against apoptotic cell death J Biol Chem.
275, 10388–10388.
34 Veijola, J., Koivunen, P., Annunen, P., Pihlajaneiemi, T & Kivirkku, K.I (1994) Cloning, baculovirus expression, and char-acterization of the alpha subunit of prolyl 4-hydroxylase from the nematode Caenorhabditis elegans This alpha subunit forms an active alpha beta dimer with the human protein disulfide iso-merase/beta subunit J Biol Chem 269, 26746–26753.
35 Takahashi, Y., Takahashi, S., Shiga, Y., Yoshimi, T & Miura, T (2000) Hypoxic induction of prolyl 4-hydroxylase alpha (I) in cultured cells J Biol Chem 275, 14139–14146.